For thirty years, the advances made in our lab in neural mechanisms responsible
for the generation of the sleep-waking cycle have been obtained by a multidisciplinary
approach and the development of new methods of investigation. Indeed,
physiological, neuroanatomical and pharmacological studies have been combined
to identify the neuronal circuits implicated in the regulation of sleep
and wakefulness. We have recently discovered a new investigational technique
that has already increased our knowledge of this neural control. Before
describing this new technique, however, some basic principles of neuroanatomy
may prove useful.

Neuroanatomy: Basic Principles

Modern neuroanatomy is almost entirely at the microscopic level. Even
the most powerful microscopes, however, by themselves are insufficient
to examine the simplest of neuroanatomical structures. Without proper
stains and staining techniques, nuclear cell groups and their connections
with other neuroanatomical sites are virtually invisible.

A classic neuroanatomical technique is to inject a "tracer" such as Horseradish
Peroxidalse (HRP) into a restricted site in the brain (1).
This substance is taken up by the axons that project to the neural structure
of interest and is naturally transported by the axon to the cell body.
Therefore any cell bodies and axons throughout the brain that are found
to contain the tracer HRP are likely to be connected to the neural structure
where the injection was made. Now HRP is chemically combined with Wheat
Germ Agglutinin (WGA), known as WGA-HRP, to greatly enhance the sensitivity
of the methods (2).

This technique would be incomplete without the ability to visualize which
axons and cell bodies in the brain contain the tracer. The most common
method to do this is known as histochemistry. Typically, the brain is
sliced into very thin sections that are washed with a solution that enzymatically
colorizes the neural elements containing the tracer. In this way, one
can localize cell bodies and axons in successive brain sections and one
can map areas of the brain that are connected to a particular neural structure.

We have recently discovered a new tracing method using cholera-toxin
instead ot the previously used classical molecules such as HRP and WGA-HRP.
In contrast to HRP, which is passively taken up by neurons, cholera-toxin
(a bacterial toxin produced by Vibrio cholerae) binds specifically to
surface receptors of neurons and is actively taken up and transported
by the axons. This phenomenon helps enhance the sensitivity of cholera-toxin
as a tracer (3)

Surprisingly, cholera-toxin chemically combined with HRP (CT-HRP) was
originally described as a sensitive tracer in the peripheral nervous system
in 1977 (4) and 1982 (5) and once
had been applied in the central nervous system (6).
Cholera-toxin was apparently abandoned as a tracer for two reasons. First,
it was found that CT-HRP was only as sensitive as WGA-HRP in the peripheral
nervous system. Second, perhaps researchers were reluctant to USC cholera
toxin because of its potential toxic properties.

Cholera-toxin is actually composed of two molecular subunits: A and B.
Subunit A is responsible for the toxic effect, whereas the non-toxic subunit
B is responsible for the internalization and transport of the toxin in
axons and cell bodies. Since the B subunit became commercially available,
we decided to try it as a tracer. We were the first to inject free cholera-toxin
(B subunit) which was not chemically bound with HRP. We were surprised
to discover immediately after the first experiment there was a much greater
sensitivity with this method relative to WGA-HRP.

The increase in sensitivity that we obtained with this tracer was not
only due to its molecular properties, but also due to the method we used
to inject it into a neural structure, as well as the staining technique
we developed to visualize it. A special injection technique known as iontophoresis,
instead of the traditional pressure injection method, permitted us to
obtain very restrictcd injection sites of less than 250u in diameter with
no necrosis of tissue providing optimal labeling, a performance never
before reached by other tracers (7). To visualize the
cholera-toxin, we developed an immunohistochemical technique in place
of the classical enzymatic "colorizing" histochemical methods used with
tracers such as HRP and CT-HRP. With immunohistochemistry, the brain slices
are washed with a solution that contains antibodies to cholera-toxin.
The antibodies bind specifically to the tracer and~ with proper staining
techniques, can be visualized under the microscope.

Summary

The discovery of this new method in 1987 has already lead us to describe
previously unknown connections to nuclei classically implicated in the
sleep-waking cycle. For example, it was previously thought that the rat
locus coeruleus (LC), a nucleus in the brain stem, implicated in sleep
wake control, received major projections from only two nuclei (prepositus
hypoglossi and paragigantocellularis lateralis). These neural connections
were described using WGA-HRP as a tracer. We recently demonstrated using
cholera-toxin that the LC also receives major projections from numerous
other neural structures (lateral preoptic area and posterior hypothalamic
areas, periaqueductal grey and nucleus Kolliker-Fuse). These results highlight
the superior sensitivity of cholera-toxin over WGA-HRP as a tracer and
indicate that the additional nuclei we described projecting to the LC
may be of crucial importance in the control of LC cell discharge and by
extension, the sleep-waking cycle. We are currently testing these hypotheses
in the lab.

By developing new technologies such cholera-toxin tracer, we may better
understand anatomy of sleep controlling centers in the brain. Neuroanatomical
mapping has already implicated anatomical structures as potential candidates
and may add another piece to the mysterious puzzle of how brain controls
sleep and wakefulness.

Pierre Luppi, Ph.D. is a neuroanatomist specializing in neural mechanisms
of sleep. Working under the direction of Dr. Jouvet for the past eight
years, he is a young upcoming CNRS researcher (equivalent to the NIH in
the U.S.A.) He received his doctorate degree neuroscience from the University
of Claude Bernard Lyon. His recent discovery of a new neuroanatomical
technique has already altered our understanding of the neural control
of wakefulness.

Figure 1 : Photomicrograph illustrating
the effects of a microiontophoretic injection of cholera toxin
in the pontine reticular formation of the cat.